Noninvasive molecular imaging methods are used to provide noninvasive dynamic information on gene expression, transcriptional regulation of gene expression, and tumor microenvironment and the consequences of chemo- and/or radiation therapy.
For example oxygen deficiency, or hypoxia, is a central microenvironmental tumor stress that arises as a consequence of the expansion of solid tumors by cancer cell proliferation, which is unmatched by the expansion and maintenance of the vasculature supply. Tumor cells surrounding functional blood vessels are generally better oxygenated, and tumor cells distant from blood vessels are poorly oxygenated. The irregular blood flow in tumors exposes tumor cells not only to chronic hypoxia, but also to acute hypoxia in regions with intermittent blood flow. Hypoxia has been recognized to induce gene instability, and also to provide an important selective pressure resulting in increased tumor aggressiveness and resistance to hypoxia-induced apoptosis. Hypoxia also leads to tumor resistance to radiation and chemotherapy treatment regimes. Hypoxia has been demonstrated in clinical trials to be associated with a poor prognosis. Accordingly, knowledge of the hypoxic state of a tumor may influence clinical treatment decisions.
Noninvasive molecular imaging methods may also be used for tracking cell location and differentiation in vivo following cellular based therapies including stem cell therapies, bone marrow transplantation, gene therapy and immune cell therapies. Such information aids scientists in biological and preclinical research, as well as providing guidance during gene therapy and in detecting, and monitoring the fate of cells during clinical treatment.
Noninvasive molecular imaging of dynamic processes has benefited tremendously from the use of reporter genes. These genes encode for proteins that emit light, bind radio-labeled probes or modulate MRI contrast. Reporter genes play a pivotal role in monitoring cell trafficking, gene replacement therapy, protein-protein interactions, neuronal plasticity, and embryonic development. To serve as a reporter gene, it is important to show not only that the encoded polypeptide can be detected in a manner that would faithfully correlate spatially and temporally with information to be gained, for example, transcription regulation or hypoxic state of a tumor microenvironment, but also that the reporter expression in the cells of interest will not alter the fate of the cells.
MRI reporter genes have the advantage that the specific signal can be coregistered with soft-tissue anatomy and functional tissue information and have, therefore, become an active and growing area of scientific interest. Several strategies exist for generating MRI contrast: using enzyme-catalyzed chemical modification of metal-based contrast agents or (phosphorus) metabolites, iron-binding and iron-storage proteins to accumulate iron as a contrast agent, and artificial proteins for imaging based on chemical exchange saturation transfer.
Current MRI reporter genes include creatine kinase, tyrosinase, β-galactosidase, transferrin receptor, ferritin, the bacterial iron transporter Mag A and a lysine-rich protein (LRP). The disadvantages of these current reporters include low resolution imaging, signal dependency on availability of iron, false signal generation, difficulties with accessibility and nonspecific uptake of substrate nanoparticles, delay of change in signal that is dependent on iron availability and ferritin loading factor, and low sensitivity.
For example, ferritin is the main iron storage and controlled-release protein in mammals, which plays a key role in the iron metabolism of mammals. Ferritin forms a highly symmetrical spherical polypeptide shell, termed a “ferritin particle”, able to store up to 4500 iron atoms as non-magnetic nanocrystal of ferrihydrite in its core (FIG. 1). Ferrihydrite is found in the core of ferritin.
Due to this paramagnetic core, ferritin exhibits magnetic properties and has recently proposed as MRI reporter gene, see for example U.S. Pat. No. 8,084,017, which is incorporated herein in its entirety. The iron moments in each ferritin core tend to align antiferromagnetically, where almost all spins cancel as pairs of aligned in opposite directions spins. Thus, net core magnetic moments (up to 300 μB) arise from uncompensated spins at the surface of the core.
However, compared to superparamagnetic iron oxides (8000-63000 μB), native ferritin has a relatively low R2 relaxivity and thus provides relatively low sensitivity as MRI contrast agent. Considering, that iron oxide form can be converted to magnetite and maghemite (γ-Fe2O3) within the ferritin core through oxygen reduction and heating (magnetite), followed by oxidation (maghemite), one way to increase sensitivity of ferritin is to convert the ferrihydrite in its core into magnetite as has been done chemically, to form magneto-ferritin.
Magnetite can also be generated biologically. Magnetotactic bacteria, which mineralize iron into a particular iron oxide, serve as an example of such process. In these microorganisms, the biomineralization of iron takes place in the magnetosome, a specialized subcellular organelle, assembled from a chain of bilayer lipid invaginations that each induce the deposition of—and enclose—a ˜50 nm crystal of magnetite (Fe3O4) or its sulfide analog, greigite (Fe3S4) (FIG. 2).
The magnetosome expresses unique sets of soluble and integral-membrane magnetosome associated proteins (MAPs) that are essential for magnetite formation. Specifically, it has been shown that Mms6 interacts directly with magnetite.
Mms6 is a small acidic MAP that contains a Leu-Gly-rich motif. Of the whole set of magnetosome-associated proteins that are linked to magnetite biomineralization, Mms6 (FIG. 4) is the only protein that has been shown in vitro to undergo proteolytic processing from its pre-protein (˜136 amino acids) to its active form (˜77 amino acids). This active component, being tightly bound to the magnetite surface, is able to interact directly with magnetite. It has been predicted that the active component of Mms6 is a peptide composed of a non-structured hydrophobic tail attached to a C-terminal α-helical portion. It has been demonstrated that Mms6 interacts with magnetite via its α-helical C-terminus. It has also been found that Mms6 deletion in vivo yielded a deformed magnetite crystal with additional crystal faces that are not present in the magnetite of wild-type bacteria.